Armed peptides

ABSTRACT

This invention relates to peptides useful for releasing active agent in the fields of diagnostics and drug delivery.

FIELD TO WHICH THE INVENTION RELATES

This invention relates to the fields of drug delivery and diagnostics.In particular the invention relates to the release of active agents frompeptides or from liposomes containing such peptides or cells containingsuch peptides in drug delivery or diagnostic applications.

BACKGROUND

Cytolytic peptides or cytolysins have previously been used to releaseactive agents or “payload” from liposomes or cells. The mode of actionfor such peptides involves perturbation of the liposome or cellularmembrane. These peptides include toxins from insects, fish, antibioticpeptides and synthetic peptides such as melittin, alamethicin,gramicidin, magainin and pardaxin, GALA, KALA, hemagglutinin subunitHA-2. Natural potent cytolytic peptides are found widely from insects tomammals, particularly as antimicrobial peptides or defensins, where theyare involved in innate defence at mucosal membranes and as cytolysins inlymphocytes. In order to target and localise the cytolytic action ofsuch peptides, a number of specific steps e.g. activating synthesis,release from lysosomes, cleavage of pro-peptides is required. Thebiological delivery activity of such peptides is tightly controlled atthe cellular and molecular levels. Biologically, cytolysin activity iscloaked by sophisticated mechanisms available within and between cellsbutthese mechanisms are diagnostically and therapeutically lessexploitable. This has therefore hindered the use of cytolysins indiagnostic and therapeutic applications.

Whilst much sought after, there are remarkably few simple and rapidhomogeneous biodetection methods.

Owing to their inferior sensitivity and non specific variablebackground, compared to the automated heterogeneous technology which isnow widespread in immunodiagnostics and high throughput screening, ithas not always been possible to develop homogeneous assays for differentanalytes. Liposomes have, previously, been utilised in homogenous assaysusing complement-mediated lysis (Anal. Biochem. 118 (1981) 286-293.)However, such assays are considered unreliable as they involve manylabile components, any one of which may become inactivated eliminatingpayload release. The development of a homogenous liposomal assay usingnon-specifically labelled digoxin melittin as the lytic agent wasreported as an alternative to the complement assay (J.Immunol. Methods.70 (1984)133-140). This method, however, has not gained widespread useas the preservation and stability of lytic activity, as well assolubility of the conjugates posed problems largely restricting the useof this cytolytic peptide to measure digoxin. This may be expected,primarily due to the uncertainties involved in the production of usefulcytolysin conjugates by relying solely on natural peptides with multiplelabelling sites, most of which are critical for peptide function and,thus, not ideally placed for retaining high activity if modified.Furthermore, the degree of modulation in activity of these conjugates isoften inadequate resulting in high background signals. Owing to thesedifficulties when using natural peptides, others have used conjugateswith a larger cytolysin, namely phospholipase C, non-specificallylabelled with analytes (J.Immunol. Methods 170 (1994) 225-231). Suchconjugates had superior solubility and greater retention of activityafter modification. However, only 75 to 85% activity was specificallyinhibited in the presence of anti-serum, which is comparable to thelevel of inhibition normally used for measuring digoxin withmelittin-oubain conjugates. A reliable assay should only permit therelease of marker molecules upon external trigger and the backgroundleakage should approach zero or at least remain constant over the assayperiod. To our knowledge neither of these conditions have beensatisfactorily addressed by homogeneous liposomal assays, withoutchanging to a heterogeneous assay configuration. Consequently, with suchassays there is always a danger of the background signal progressivelyinterfering with the analyte dependent signal. Some of the long termbackground problems arising from the use of liposome reagents per se canbe overcome by the use time resolved fluorimetry, in which a largermolecular weight protein chelator conjugate is encapsulated in theliposomes, allowing fluorescent detection upon cytolysin mediatedcomplexing with ions such as Eu³⁺(Anal. Biochem 238 (1996) 208-211).Even with these assays, the inherent problems of the non-specific lysisby uninhibited conjugates as well as optimising conjugates to produceadequate activity, remain. Consequently, such assays need to beperformed under well-controlled laboratory conditions and at fixed timesagainst the varying background signal.

Liposomes have been used more widely in drug delivery rather than indiagnostic applications and or as imaging agents, however, in all casesthere has been little progress made with the use of liposomes, effortsbeing mainly devoted to developing different lipid formulations to tryto achieve controlled and quantitative release of active agent orpayload in response to a trigger.

For a reliable assay, the release of detectable marker molecules shouldonly occur in response to an external trigger and any leakage of markermolecules should be minimal for example, approaching zero, or at leastremain constant over the assay period. Consequently, in such assaysthere is always a danger of background signal or interference caused bythe progressive release of marker molecules.

Our earlier patent application WO98/41535 (PCT/GB98/00799) describespeptides which can be efficiently cloaked and used to release a“payload” in a controlled manner. The peptides disclosed in thatapplication were non-responsive to pH change particularly over a narrowrange between pH 6.5 and 7.4. On the contrary, in most cases, loweringof the pH would result in the lowering of peptide activity. A number ofpH sensitive peptides have been used to release payload from liposomesunder acidic conditions (Advanced Drug Delivery Reviews 38 (1999)279-289). For these peptides the triggering range is, however, far fromphysiological pH, usually requiring pH values lower than 6 to releasepayload from liposomes.

GALA is one of the most efficient pH specific peptide. For this peptideCalcein release from liposomes has been demonstrated at values lowerthan 6. There are many other pH specific peptides, such as Influenzavirus HA-2 N-terminal peptide, EALA, JTS1 and Rhinovirus VP-1 N-terminalpeptide which have been shown to release liposome contents in low pHenvironments such as the endosome where the pH is reported to be wellbelow 6 and typically 5. There are several pH sensitive peptides knownin the literature to destabilize liposome membranes. However they areusually triggered at very low pH (5.5) and consequently have foundlittle or no use in drug delivery, for example, to tissues or tumourswhere the pH difference between normal and diseased areas can be lessthan a one pH unit. Their major use thus remains endosome delivery.

The strategy of micro-environmental pH change in tissues to inducepreferential release of drugs from liposomes requires peptides torespond over a narrow pH change, closer to the physiological range. Toour knowledge there are no reported peptides which trigger release ofpayload from liposomes efficiently and close to physiological pH levelsof 7.4 while their background biological activity remains low or zero ator close to pH 7.4.

A peptide named “helical erythrocyte lysing peptide” (HELP) (ProteinEng. (1992), 5, 321) is known to lyse red blood cells and has been shownto trigger release of haemoglobin below pH 6.5 only. This peptide is,however, specific to lysing cells and there are no reports showinglysing of liposomes. We have shown that liposomes could not be lysed ina pH specific manner using this peptide.

WO97/38010 relates to fusogenic liposomes and delivery systems fortransporting-materials such as drugs, nucleic acids and proteins. Thesesystems work by fusion of liposomes and at pH values lower than 6.

DESCRIPTION OF THE INVENTION

According to one aspect of the invention there is provided a cytolyticor agent delivery peptide, wherein the cytolytic or agent deliveryactivity of the peptide is modulated by changes in one or moreparameters which directly or indirectly affect the peptide, whereinchanges in one or more such parameters leads to cytolysis or agentdelivery by the peptide at a pH close to physiological values. Theinvention therefore provides a “cloakable” cytolytic or agent deliverypeptide whose activity can be harnessed and maintained low by arming buttriggering only with another parameter or stimuli. The invention findsuses in in vitro, in vivo diagnostics, and in the delivery and targetingof drugs. Preferably, the cytolytic or agent delivery activity ismodulated by changes in more than one parameter. More preferably, onesuch parameter is pH.

According to another aspect of the invention there is provided acytolytic or agent delivery peptide, where the cytolytic or agentdelivery activity is modulated by a change in pH, from a starting pH toa modulating pH where the starting pH is close to physiological pHvalues. Preferably the pH value is less than 7.40. Thus in certaindisease conditions in which a change of pH occurs as cells go from anon-diseased to a diseased state, an active agent can be released by thepeptides in response to that pH change.

The parameter may be for example pH, the effect of a ligand e.g for areceptor or enzyme, temperature, light, ultrasound; redox potential,DNA, nucleic acid binding, or binding of the peptide to liposome, toform a non-leaky complex i.e. one where the active agent or payload isnot released by the liposome until delibrately triggered. pH is apreferred parameter.

The peptides are designed to increase in hydrophobicity as the pHdecreases from neutral to slightly acidic while retaining substantialpositive charge. The prior art pH sensitive peptides (Parente et al,Biochem, (1990) 29, 8720); Subbarae et al, Biochem (1987) 26, 2964-2972)have predominantly Glu residues (pK_(a) about 5) and cannot fulfill therequired pH sensitivity for triggering closer to neutral. We find thatthe negative character can be counterbalanced by carefully includingbasic residues into the sequence resulting in desired pH sensitivity.Similarly the hydrophobic character can be counterbalanced by includingfatty acid or modifications containing alkyl chains carefully positionedin the sequence. Suitable modifications include myristoyl, palmitoyl,dioleoyl, phosphoplipid, farnesyl, undecyl, octyl and geranyl. Thehydrophobic-anionic-cationic character of a peptide is a crucial factorin achieving a narrow triggering range on setting closer to neutral butoff setting at physiological pH. The triggering range can be tuned towithin the 6.5 to 7.4 pH range.

Examples of the peptides of the invention are given in Table 1. Many ofthe peptides in our table 1 could be modified to producemulti-triggering properties. For instance peptide 13 in particular couldbe phosphorylated on Ser to bring about inactivation (like peptide 12)and it could be biotinylated on Lys to inactivate with avidin binding(as in peptide 1) and it could in addition be inactivated by DNA bindingon C terminal. Thus if desired its pH triggering properties do not needto be utilised.

In one embodiment, the invention provides a pH sensitive cytolyticpeptide, having a cloaking site, and which is integrated with or canintegrate with a lipid vesicle and can be activated closer tophysiological pH in order that antibody or receptor binding at thecloaking site is near optimum while its activity at physiological pH canbe harnessed and maintained low by control of pH levels affecting thepeptide. In a preferred embodiment the integration with liposomes isachieved by covalently linking a hydrophobic group such as a fatty acidonto the peptide. There are several other lipids such as palmitic acidor isoprenyl groups which could also be used to conjugate liposomes withpeptides. Further ways of peptide incorporation include: covalentlinkage to phospholipid, binding to receptors or ligandspre-incorporated on liposome surface or cells, encapsulation of thepeptide in the liposomes, attachment of hydrophobic or amphiphilicsegments to peptides and electrostatic binding to charged membranes.

The dual switch feature embodied in peptides of the invention haspotential uses in drug delivery systems.

For example, in the case of a pH-responsive cytolytic or agent deliverypeptide, the peptide could be maintained in an inactivated form by aproteolytic sensitive sequence, the peptide would then be activatedafter proteolytic cleavage and when the pH conditions are met (usuallyacidic).

For instance the cloaking site can be phosphorylated and triggeringwould then involve dephosphorylation and pH change. Alternatively, thecloaking site may be modified with a proteolytic sensitive sequence,cleavage of this sequence then activating the peptide.

In the case of a pH-responsive peptide, the peptide could be inactivatedby DNA binding, the peptide would then be activated after DNAdissociation and when the pH conditions are right (usually acidic).

A pH-responsive peptide could initially be inactivated by antibodybinding and then activated in presence of an analyte or an epitope andwhen the pH conditions are right (usually acidic).

A pH-responsive peptide-liposome complex could be targeted by theinclusion of a cell-specific sequence to particular cells in aninactivated state and adapted so that the microenvironment of the cellsactivates the peptide to release payload.

A pH sensitive peptide could include a specific sequence so that it canbe targeted to cells in an inactivated state, triggering then beingeffected with external stimuli or by addition of pH modulator. Examplesof pH modulation include glucose-mediated decreases of pH in somemalignant tissues and bolus injection of MIBG (meta-iodobenzylamine)with glucose resulting in pH activation to release payload from thepeptide. There are many examples of cell targeting sequences.Endothelial cells in angiogenic vessels within the tumour have markerssuch as alpha integrins. The motif Arg-Gly-Asp (RGD) present in peptidestructures binds selectively to integrins. Similarly there are severalother motifs such as NGR, GSL. The screening of phage display peptidelibraries is greatly enhancing discovery of new targeting sequences. Forinstance, several prostate-specific antigen (PSA) binding peptides areknown.

The peptide may have continuous stretches of basic and acidic sequencesand trigger close to physiological pH to effect lysis of biomembranes orcondense/decondense DNA closer to physiological range. Preferably a pHsensitive peptide comprises a highly basic sequence at one end and ahighly acidic sequence at another end and the overall Pi value liesbetween 6.4 and 9.

In peptides in accordance with the invention, changes in one or moreparameters leads to cytolysis or agent delivery by the peptide at a pHclose to physiological values.

In particular, the pH value at which the cytolytic or agent deliveryactivity occurs may be less than 7.40, preferably between pH 6.5 and7.4; pH 6.6 and 7.4; pH 6.7 and 7.4; pH 6.8 and 7.4; pH 6.9 and 7.4; pH7.0 and 7.4; pH 7.1 and 7.4; or pH 7.2. and 7.4.

The hydrophobicity of the peptide may increase as pH decreases whilstretaining a substantial positive charge.

The cytolytic activity or agent delivery activity may include releasingan agent which has been bound to the peptide.

The peptide may have a predominantly negatively charged portion with arelatively low Pi value and a predominantly positively charged portionwith a relatively high Pi value. The negatively charged portion maycontain at least two amino acids having a relatively low Pi value. Thesaid at least two amino acids may be selected from glutamine acid andaspartic acid.

Preferably the Pi value of the negatively charged portion is about 4.

The positively charged portion contains at least two amino acids with arelatively high Pi value. The Pi value of the positively charged portionmay be about 9. The said at least two amino acids may be selected fromlysine, arginine or histidine.

The positively charged or negatively charged portions may be at or nearthe ends of the peptide or provided by side chains of the peptide.

The negatively charged portion may have the composition:X_(n1) Y_(n2)

where n1 is ≧2; and

n2 is 7−n1

and where X=glutamic acid and/or aspartic acid

Y is any amino acid other than lysine, arginine or histidine.

The positively charged portion may have the composition:A_(n1) B_(n2)

where n1≧2; and

n2=Z−n1

and where X is glutamic acid and/or aspartic acid

B is any amino acid other than glutamic acid or aspartic acid

Z=any integer from 7 to 14.

Preferably Z=9, or 7. Most preferably Z=9.

Other aspects of the invention are apparent from the appended claims.

A peptide can be used to form complexes with liposomes without payload.The complex can then be used to load agents or “payload” into liposomesat an acidic pH the payload then being trapped by raising the pH.

An armed peptide can be encapsulated into liposomes whereby lysis of theliposomes takes place from within the liposomes by unarming the trigger.The trigger can be unarmed by, for instance, proton movement, in case ofa pH-responsive peptide, which can be aided or unaided by protonophoresor by cofactors, in case of an enzyme sensitive sequence or by otheractivation means.

According to another aspect of the invention there is provided a methodof releasing an active agent from a lipid vesicle the method comprisingaltering more than one parameter relating to a cytolytic peptide wherebythe cytolytic peptide is activated to cytolyse the lipid vesiclecontaining the active agent.

The lipid vesicle may be for example a cell or a liposome.

By using more than one trigger to cloak cytolytic peptides in diagnosticmethods according to the present invention, the background signal can beimproved and maintained low and stable allowing simpler assayconfigurations and more predictable development for multiple ofanalytes. This invention provides peptide-liposome complexes whichtrigger close to physiological conditions. The complexes can respond toslight variations in physiological pH. Further aspects of the inventionrelate to controlling triggering of peptides or their liposome complexeswhich can be effected by altering at least two or more parameters one ofwhich is used to arm the release of payload as a “safety catch”. In thepreferred embodiment the peptides are integrated with liposomes ascomplexes.

Definitions

The following definitions are given by way of explanation:

The term “peptide” used in this specification embraces polypeptides andproteins formed from natural, modified natural and synthetic aminoacids.

The term “cytolysis” means the disruption of particles such as cells,liposomes, biomembranes or polymers.

The term “active agent” includes non-biologically active substances suchas imaging agents or fluorophores.

The term “payload” means anything encapsulated in a liposome and whichcan be released by the peptides of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

Peptides, peptide/liposome complexes, methods of drug delivery,diagnosis in accordance with the invention will now be described, by wayof example only, with reference to the accompanying drawings, FIGS. 1 to15, in which:

FIG. 1 is a schematic illustration of the concept of armingpeptide-liposome complexes in accordance with the invention;

FIGS. 2A and 2B are graphs illustrating the detection of biotin by pHarming peptide 1 (table 1);

FIGS. 3A, 3B and 3C are graphs and 3D is a photograph illustrating anexperimental biotin assay with peptide 1 from table 1;

FIG. 4 is a graph showing the effects of different pH levels in therelease of dye from liposomes with various peptides from table 1.

FIG. 5 is a graph and shows the results of experiments involving bothantibody binding and pH arming at pH 7.4.

FIG. 6 is a graph illustrating the results of experiments with an assayfor the detection of VTB epitope;

FIG. 7 is a graph illustrating the results of experiments with an assayfor the detection of VTB subunit;

FIG. 8A to F are graphs illustrating the release of dye from peptideliposome complexes at different pH levels;

FIG. 9 is a graph showing the results of testing the complex of peptide1 tested 40 minutes from preparation;

FIG. 10 is an image of a Rif tumour after administration of liposomesand pH armed peptide liposome complex; and

FIG. 11 is a graph showing the release of dye from liposomes in atumour;

FIG. 12 is a graph showing the release of dye from liposomes in atumour;

FIG. 13 is a graph showing the release of dye from liposomes in atumour.

FIG. 14A is a graph showing lysis of calcein liposomes with peptide 12in the presence (upper curve) and absence (lower) of alkalinephosphates.

FIG. 14B is a graph showing activation of a peptide by an enzyme(alkaline phosphatase) giving calcein release at acidic pH. Curve 1represents the enzyme treated peptide whilst curve 2 representsnon-treated peptide; and

FIG. 15 is a graph showing relative lysis of calcein liposomes at acidicand physiological pH in the presence and absence of DNA.

Competitive reactions could be used, particularly when the affinity orimmuno-specific trigger is armed by another mechanism (e.g., pH), whichcould also be expected to improve the practical fidelity of adisplacement trigger. A pH sensitive peptide of sequenceMyr-EAALAEALAEALAEGK*PALISWIRRRLQQ-anide was designed, modified withbiotin at cloaking site (*) and integrated with liposome.

This peptide-liposome complex exhibits pH dependent activity dependingon concentration of peptide used. The modified peptide retained itsactivity and pH responsive properties upon modification with biotin. Atacidic pH the peptide-liposome complex is active while at alkaline pHthe complex remains inactive (FIG. 2). In FIG. 2, the curves from top tobottom represent (i) Peptide at pH 6.6, (ii) Peptide at pH 8 and (iii)background signal in the absence of any peptide. In FIG. 2A, the curvesfrom top to bottom represent peptide in the presence of (i) 11 piccolosof biotin and 1,2 fold excess avidin at pH 6.6, (ii) 1.2 fold excessavidin at pH 6.6, (iii) 11 piccolos of biotin and 1.2 fold avidin at pH8 and (iv) 1.2 fold excess avidin at pH 8. Further the complex could beefficiently cloaked with avidin binding. Data in FIG. 2 reveals that inthe presence of analyte (biotin) detection could be triggered only atacidic pH while only slight release was noted at pH 8 thus providingevidence for dual switched peptide where both the affinity reaction andpH are required to release liposome contents.

The biotin assay using pH sensitive peptide was repeated in a glass vialwhich could be illuminated with a small torch or blue LED light source.Specifically, the vials contained 1.2 ml of biotinylated hybrid peptide(17 nM) in PBS buffer pH 6.2 containing calcein liposomes (4 mM lipid)and 1.2 fold excess avidin. Biotin was present in vials 3 to 6 inincreasing concentration. Vials are as follows: (1) Background samplecontaining liposomes only, (2) no biotin, (3) 12 pmoles biotin, (4) 15pmoles biotin, (5) 17 pmoles biotin, (6) 50 pmoles Samples wereilluminated by simple 3 mm blue diodes from underneath the cuvettes.Photographs in the order top to bottom, were taken at indicated timesfrom addition of liposomes. The actual fluorescence readings taken on afluorimeter at five minute period are also shown graphically. Fiveminutes after the addition of all reagents the fluorescence becameclearly visible to the naked eye compared to the background whichremained low and constant allowing detection of 12 picomole of biotinrapidly without any instrumentation. (FIG. 3). However, instrumentationavailable in most laboratories (e.g., fluorescence, absorption,luminescence, electrochemical, biosensors), can also be used forquantitative analysis by changing the signal development moleculesincorporated into the liposomes.

The general concept of using eloaked cytolytic peptides with biospecifically switched activity is illustrated schematically in FIG. 1.Structural and covalent modifications at the cloaking site preventsaction of the peptides on biomembranes by one or more mechanisms (FIG.1). These may include pH, ligand, steric hindrance, (e.g., antibodies,avidin) and redox titration of ionisable moieties. When this is reversedby back titration of the ionisable groups, by release of the boundprotein or by enzymatic cleavage of the cloaking moiety, biomembranescan be permeabilised to small and large molecules. This can be used forthe controlled release of diagnostic or therapeutic payloads fromliposomes or may be applied directly to permeabilise cells. The use ofmore than one trigger to activate the peptides would be expected toimprove the fidelity or specificity of the process, and, in principle,any combination could be used. For example, the use of aphysiologically-relevant physicochemical activation (e.g., pH, redox)combined with an immunospecific trigger may be used to control cytolysinaction on cells, to release amplifiers from liposomes for sensitivediagnostic tests on specific viable micro-organisms and for thebioresponsive release of drugs from liposomes.

Integration of peptide assemblies into lipid bilayer can be driven byintroducing non charged terminal to form tethering site while cloakingsite is regio-specifically located at position near the central part ofpeptide most likely to affect its function quantitatively by bindingreactions. Line is drawn to show the polar-apolar interface. Mechanismsinvolved in activating peptides could include steric, pH, redox, enzymiccleavage or a combination for multi-triggering with the result thatdissociation of the cloaking molecule and presence of trigger frees thecytolytic peptide to breach the membrane. In the schematic above peptideis allowed to form complex with liposomes (lipid bilayer shown) whilecloaked with receptor. The uncloaked peptide although liposomeassociated remains inactive until another parameter such as pH isaltered and peptide becomes active to cause lysis. It is obvious tostate that cloaking can be done either before or after liposome complexis formed to function in competitive or displacement modes.

EXAMPLES

The primary sequence of the peptides are given in table 1. The cytolyticpeptides were manually synthesised by solid phase t-Boc chemistry using0.5 mmol of p-methylbenzhdrylamine (MBHA) resin. Side chain protectionsfor amino acids (BACHEM UK) were 2-chlorobenzyloxycarbonyl for Lysine;p-toluenesulfonyl for Arg, Benzyl for threonine, serine and glutamicacid. Couplings were made using 1.5 mM amino acid, 1.5 mMolesbenzotriazol-1-yl-oxytris (dimethylamino) phosphoniumhexafluorophosphate (BOP) and 4.5 mM N,N-diisopropylethylamine (DIPEA)in N,N-dimethylformamide (DMF) for 40 mins. Second coupling was usedwhen necessary to drive the reaction to almost completion (>99.8%).Myristic acid was coupled in same manner as an amino acid. In the caseof peptides where the sequence was branched off from Lysyl residue(sequences shown in brackets in table 1) we used Fmoc protection on theε-amino which was selectively deprotected using 20% piperidine andsynthesis continued as usual. At the end of synthesis the peptide wascleaved using HF in the presence of 0.5 g p-thiocresol and 0.75 gp-cresol as scavengers. The peptides were purified on a C-4 reversephase semi-preparative column (Vydac C-4, 250×4.6 mm) using anacetonitrile/0.1% TFA gradient. The HPLC purity of the peptides wasdetermined by analytical reverse phase HPLC. No peptides were used below95% purity level. Characterisation was made using MALDI(Thermobioanalysis) mass spectrometry.

Table 1 peptideshows examples of peptides which were synthesised andwhich were shown to trigger in slightly acidic buffers whilst exhibitingvery low or no activity at pH 7.4.

TABLE 1 Chemically modified peptides SEQ ID NO: Sequence 1.Myr-EAALAEALAEALAEGK(biotinyl) PALISWIRRRLQQ-amide 2.Myr-EAALAEALAEALAEGKPALISWIRRRLQQ-amide 3.Myr-EAALAEALAEALAEGKPALISWIRRRK(myristoyl) QQ-amide 4.Myr-EAALAEALAEALAEGKPALISWIIQQK(myristoyl)- amide 5.Myr-EAALAEALAEALAEGKPALISWIRRLQQ-amid 6. Myr-EAALAEALAEALAEGK(ELFTNR)PALISWRRRLQQ-amide 7. Myr-GIGAVLRVLTTG(TLLEFLLEELLEFL)KPALISWIRRRQQ-Amide 8. Myr-EAALAEALAEALAEGKPALISWIRRRQQ K(Myr)- Amide 9.Myr-WEAALAEALAEALAEHLARALAEALEALAA- Amide 10.Myr-WBALAEALAEALAEHLAKALAEALEALAA- Amide 11. Myr-GIGAYLRVLTTG(TLLEFLLEELLEEL)KPALISWIRRRRQQ-Amide 12. Myr-WEA ALA EAL AEA S(Phospho) AE HLA RALAEA LEA LAA-Amide 13. Myr-LEAALAEALEALAAGKPALISWIRRRRQQ- Amide

For preparing biotinylated peptides the peptide (0.02 mmole) wasdissolved in 4 ml DMF and Biotin N-hydroxysuccinimide (0.1 mmole) addedfollowed by DIPEA (0.3 mmole) and the mixture stirred. In all thesepreparations the reaction was allowed to proceed until completion asjudged by the decline in amine content using a ninhydrin assay. Thesolvent from reaction mixtures was removed under vacuum and the productwas purified by reverse phase HPLC on a C-4 preparative column usingacetonitrile and 0.1% TFA gradients. Characterisation was made by massspectrometry as above.

Liposomes with Payload

Liposomes encapsulating calcein dye were prepared by an extrusion method(Biochim. Biophys. Acta, 812 (1985) 55). Phophatidylcholine (50 mg) andcholesterol (13.08 mg) which had been dissolved in 4 ml of 50% v/vchloroform methanol solution were evaporated to form a lipid film in around bottom flask. If it is essential to follow the fate of liposomes alipohilic-dye such as DiI (50 μg) could be incorporated into the lipidfilm prior to hydration. The film was then hydrated with 4 ml of 120 mMcalcein solution prepared in 10 mM sodium phosphate 20 mM Sodiumchloride buffer pH 7.4. Liposomes were formed by 10 extrusion cyclesthrough 0.2 micron or 0.1 um polycarbonate filters using the Liposofast100 (Avestin) extruder device. The non encapsulated dye was removed bygel filtration on a PD-10 column using iso-osmotic buffer. The totallipid concentration of the liposomes was measured by the Stewart assayand adjusted to 3 mg per ml.

Closer to Physiological pH Switching Properties

The cytolytic activity of pH-responsive peptides was followed by adding2 or 3 μl of liposomes to a 2 ml assay volume and continually recordingfluorescence. Buffers were made of 10 mM Na phosphate, 140 mM NaCl, 1 mMEDTA, 5 mM HEPES at several different pH values. The pH profiles of thevarious peptides from table 1 peptideshowing triggering around pH 7 areshown in FIG. 4. Specifically, FIG. 4 shows the results of peptides 2, 7and 9 acting on liposomes. In the experiments 2 ml of 10 mM Naphosphate, 140 mM NaCl, 1 mM EDTA, 5 mM HEPES buffer (at pH valuesindicated on the figure) containing liposomes (4 μM lipid) and (A) 14 nMpeptide 2, (B) 20 nM peptide 7 (C) 45 nM peptide 9. In all casespeptides were added indicated by sharp dip in fluorescence were used.The pH values are as indicated for each trace. Note that this pH isclose to optimum for most receptors and such triggering has never beendemonstrated before. In general, it is well documented that manyproteins including receptors, enzymes and antibodies has pH optimumusually closer to physiological value. On either side of the pH optimumthe binding activity is reduced. However in general as activity profileis typically bell shape most proteins would tolerate at least a pH unitshift from optimum. Obviously the closer the results are to pH 7.4 thehigher chances their are for efficient binding with target proteins orreceptors.

Detection of Biotin by pH Arming

Peptides were prepared at concentrations ranging from 1 to 0.01 mg/ml,depending on the activity of peptide, in deionised. A stock solution ofavidin (5 unit per ml, one unit of avidin binds 1 μg of biotin.) wasprepared in PBS pH 7.4 buffer. Biotin, from a stock solution of 10 mg/mlprepared in DMSO, was diluted 10000 fold with water to obtain workingconcentrations of 1 μg/ml. A typical cytolytic assay was performed in atotal volume of 2 ml PBS buffer containing calcein liposomes (5 μMlipid) as prepared above. The progress of dye leakage was continuallyfollowed using excitation and emission wavelengths of 490 and 520 nmrespectively. Peptide of known concentrations-was added at certain timepoints and the solution was rapidly mixed while continuing to measuresignal. For uncloaking the peptide activity using biotin, the additionswere made to the buffer sequentially in the order avidin, 2 minuteincubation with biotin followed by the addition of biotinylated peptide(44 nM). The mixture was incubated for filter 2 minutes and fluorescencemeasurement initiated. At selected time points liposomes (7 μM) wereadded and solution mixed. For the cloaking experiments the additionswere essentially the same except no free biotin was added. The avidinconcentration was a 1.2 fold excess units to ensure complete cloaking.For evaluating dual trigger switching properties of biotinylated peptidethe liposomes (7 μM lipid) in 2 ml of buffer were treated with peptide.(2.8 nM) and fluorescence measured continually. For cloaking experimentsand pH arming the peptide solution (2.8 nM) was incubated with a 1.2fold excess of avidin solution for 3 minutes and the cytolytic assayperformed at pH 6.6 and 8. For the uncloaking experiments the avidin waspre-incubated with 11 picomoles of biotin solution. Data is shown inFIG. 2.

Visual Detection of Biotin Analyte

In the control sample, liposomes (4 μM lipid) were added to 1.2 mlsolution of peptide (14 nM) pre-incubated with a 1.2 fold excess avidin.Test samples contained biotin at known concentrations. Additions weremade sequentially in the order, biotin, avidin, followed by a 1 minuteincubation, peptide followed by two minute incubation and finallyliposomes. The samples were illuminated from underneath with simple 3 mmwide angle ultra bright blue diodes (RS Components) powered by a 3 Voltbattery. Photographs were taken with a standard digital camera after 5minutes, 1 hr and 18 hrs to visually observe the signal. The actualfluorescence readings after 5 minutes were also recorded using afluorimeter.

Detection of VTB Epitope and VTEC by pH Arming Liposomal Assay

To show the benefit of dual trigger detection the peptide Peptide 2which has pH responsive profile as shown in FIG. 4 was modified at thecloaking site with a short sequence (ELFTNR) known to be epitope ofverotoxin subunit B (Infection & Immunology (1991) 59,750-757) to obtainpeptide 6. This peptide was also pH sensitive analogous to its parentsequence Peptide 2. For the detection of VTEC at a pH where peptide isactive the conditions of the assay were: 2 ml of assay buffer (140 mMNaCl, 10 mM sodium phosphate buffer containing 5 mM HEPES and 1 mM EDTAat pH 6.8)+3 μl of calcein liposomes (100 nm diameter) were treated with10 μl of peptide 6 (Screening grade) preincubated (2 mins) withanti-epitope antibody (6 μl of mg/ml protein A pure). FIG. 5 shows thedata. The lower curve shows the same experiment at pH 7.4 in presence ofantibody. Fluorescence recorded by monitoring emission at 520 nm afterexcitation at 490 nm. The total release of calcein was achieved by theaddition of Triton X-100.

The background apparent at acidic conditions (middle curve) could bemaintained low at physiological pH values until measurement was requiredas shown in FIG. 5. It is thus clear that detection would only bepossible below physiological pH. The following examples show that thepeptide liposome complexes can be unarmed and analytes detected at pH6.8.

VTB epitope could be detected by release of calcein from 3 μl ofliposomes (100 nm) by peptide 6 in the presence of free epitope (ELFTNR)competing for anti-epitope antibody. The assay was performed in 2 ml ofbuffer (140 mM NaCl, 10 mM sodium phosphate, 5 mM HEPES and 1 mM EDTA).Peptide 6 and free epitope were allowed to compete for 6 μg antibodyprior to addition, This data is shown in FIG. 6.

VTB subunit could also be detected similarly using following conditions.Release of calcein from 3 μl of liposomes (100 nm) by peptide 6 in thepresence of VTB subunit competing for anti-epitope antibody. VTB andantibody were preincubated for 3 minutes before the addition of peptide6. The assay was performed in 2 ml 140 mM NaCl, 10 mM sodium phosphatebuffer pH 6.8 containing 5 mM IEPES and 1 mM EDTA with detectedconcentrations of epitope indicated on the trace. Data is shown in FIG.7 with detected concentrations of VTB indicated on the trace

Liposome-Peptide Integral Complex:

A fatty acid incorporated on the N-terminal of peptide anchors thesequence with liposomes to form integral complex which can then be usedas single stable reagent that can be triggered to release thecontents-of the liposomoes when the pH is ideal (i.e physiological 7.4)or acidic (e.g 6.2). In the first instance we prepared theliposome-peptide complex at a predetermined ratio of lipid-to peptide(30:1) in pH 8 buffer using peptide 10. The ratio was pre-determined bycarrying out series of lytic profiles at different concentrations and pHvalues to reach conditions whereby little or no lysis was observed at pH7.4 while significant release was evident at acidic values. The complexbetween liposomes and peptide was formed by adding peptide to 2001 μl of10 mM NaP containing 140 mM NaCl, 1 mM EDTA, 5 mM Hepes pH 8 buffer,containing 100 μl of calcein encapsulating liposomes. The mixture wasincubated for 20 mins to form the complex prior to use. In order toascertain that the lysis is occurring due to the formation of complexand not as the action of the peptide per se it was essential to purifythe liposome-peptide complex. The peptide to lipid ratio in this complexis 1:30. The complex was applied to a Sepharose CL-6B column. Fractionscorresponding to liposomes were collected as clearly visible to the eye.Aliquots of these fractions were then tested for lytic activity atacidic and physiological pH values. The key aim was to establish thatthe peptide is liposome associated and would thus be eluted withliposome fraction in the void volume. We used 100 μl complex (a columnpurified fraction) and followed the release of Calcein at two differentpH values.

FIG. 8 Shows:

FIG. 8A. Trigger of complex after sepharose CL-6B column in bufferSpecifically, 100 μl Liposome+peptide complex after elution throughSepharose-6B column equilibrated by NaP buffer at pH 8.01 was added to afinal volume of 2 ml NaP Buffer (10 mM Sodium Phosphate+150 mM NaCl+5 mMHepes+1 mM EDTA) at two different pH concentrations. Upper curve pH 5.8.Lower curve pH 7.4.

FIG. 8B Purified Peptide(P9)-Liposome Complex Conditions

2 ml buffer (10 mM Na phosphate, 140 mM NaCl, 1 mM EDTA, 5 mM HEPES)+50μl purified complex. Lipsomes used were 50 nm extruded. Curves top tobottom are pH 6.2, 6.4, 6.7, 7.0, 7.4

FIG. 8C. Lytic Assay of Complex

Complex 20 μl liposomes+60 μl pH 8 buffer+20 μl peptide (0.1 mg/ml)prepared and used within 2 minutes. 10 μl was added to each of thecuvettes containing 2 ml buffers. Top curve pH 6.2 (triton was addedtowards the end indicated by sharp dip in fluorescence to check fulllysis), the lower curve is at pH 7.4. Samples measured simultaneously.

FIG. 8D Stability of Complex (Used for in Vivo Studies) 20 Minutes fromPreparation

5 μL of complex in 2 ml of buffer tested 20 minutes after preparation.Upper curve pH 6.2, Lower curve pH 7.4.

FIG. 8E Stability of Complex (Used for in-Vivo Studies) 1.5 hr fromPreparation

5 μL of complex in 2 ml of buffer tested 5 hrs after preparation. Uppercurve pH 6.2 and lower curve pH 7.4.

FIG. 8F Stability of Complex (Used for in-Vivo Studies) 24 hrs Minutesfrom Preparation

5 μL of complex in 2 ml of buffer tested 24 hrs after preparation curvepH 6.2, Lower curve pH 7.4.

Data in FIG. 8A shows that the liposome-peptide complex remains intactand shows pH responsive properties. During purification of the liposomeswe noted some Calcein on top of the column. This indicates that there issome leakage of the dye when complex is formed. Upper trace at pH 5.8peptide shows the acid triggered release while the trace at pH 7.4 showslittle or no release indicating a stable complex.

Complex formed by another peptide (peptide 9) also showed (FIG. 8B) thattriggering properties are retained after purification. For this peptidea different peptide to lipid ratio (1:300) was used.

From the above experiments it was conclusive that the peptides remainliposome-associated to cause release of payload.

The data in the above traces was for regular Calcein liposomes. For invivo applications we incorporated another dye DiI into liposomes toassist quantification. These liposomes showed adequate triggeringproperties with the peptides. The data in FIG. 8C shows triggering ofthe peptide 9 (table 1) with these liposomes. Using several trial anderror ratios of liposomes, peptide and buffer the most optimised complexrequired 20 μl liposomes, mixed with 60 μl pH 8 buffer to which 20 μl ofpeptide (0.1 mg/ml) is added. The traces in FIGS. 8D, E, F showsstability of the complex by retention of the pH triggering of propertiesas tested 20 minutes, 5 hrs and 24 hrs after preparation.

Stable complexes without purification can be produced by optimisingpeptide to lipid ratio and concentration highlighting the reality ofproducing single reagent formulation.

Other peptides can also form complexes which trigger in acidic media(upper curve). For instance shown in FIG. 9 is the data for peptide 1.

Alternatively the complex formed could be gel-filtered to removeunattached peptide and an aliquot of eluant tested to show activity. Theformation of complexes requires a careful study of lipid to peptideratio. However once conditions are determined, the armed complexes ofthis type can be scaled up and used to release payload at acidic regionssuch as tumour. The binding of avidin to the biotinylated peptide couldalso be used to switch off the activity for added fidelity. It is alsopossible to accumulate these complexes at target sites and then modulatethe pH to release payload locally. Methods of modulating pH havepreviously been reported (Cancer Res 1982, 1505-1512 & Cancer Res 1994,3785-3792).

Similar methodology could be used for in vivo imaging of tumours wherebythe payload is a marker or diagnostic reagent sequestered insideliposomes. For instance, when using the dye calcein, the pH released dyewould be detected by increase in green fluorescence. FIG. 10 shows datato illustrate this effect with pH armed complex of the peptide withcalcein liposomes. Complex was prepared from biotinylated peptide (1) byadding 10 μl of pH 8 buffer to 41 μg of peptide to which was added 150μl of Hposomes encapsulating calcein. The complex was incubated brieflyand 100 μl was injected via the tail vein into mice with implantedtumours. The control mice were given equivalent levels of untreatedliposomes. Using the dorsal window chamber model (Biophysics.1997,1785-1790 & Nature Biotechnology 1999,17,1033-1035,) with implantedtumours (Rif-1 allografts) direct in vivo examination of the calcein inand around the tumours was made by fluorescence microscopy and computercontrolled time-lapse images. Photographs shown in FIG. 10. illustratethat the armed complex liposomes shows an intense image (B) relative tothe control liposomes (A) indicating that the complex has been unarmedby the tumour pH. Note that many tumours in animals and human, Rif-1being one example, have an ambient pH that is slightly lower than thatof normal tissue (Science 1980, 210,1253-1255 & Cancer Res1989,4373-4384). This pathophysiological feature of tumours may be usedfor cancer detection. Thus unarming the complex which triggers below butclose to physiological pH causes the release of dye almost immediately.This way the armed complexes can be used for in vivo detection ofdisease and particularly for the detection of cancer. Similarly, if thepayload was combination of calcein dye and anticancer drug it would bepossible to detect and treat with same formulation offering majoradvantages for diagnosis and treatment.

Phoshorylation of the Peptide or Complex:

The peptide or complexes of the invention can also be armed by includingsequences or chemical modifications that can be cleaved by enzymes. Thisis illustrated using phosphorylated peptide. The peptide or complexescan also be phosphorylated to achieve inactivity. De-phosphorylation andlowering of pH then provides controlled release of payload.

Peptide 12 (Table 1) was synthesised manually by solid phase Fmoc(9-fluorenylmethoxycarbonyl) chemistry using 0.25 mmole of Rink amideresin as the solid support. The following standard Fmoc-amino acid sidechain protections were used: Glu: t-Bu; His: trityl; Arg: Pmc. Forserine Fmoc-O-benzyl-L-phosphoserine obtained fromCalbiochem-Novabiochem (UK) was used. The Fmoc protections were removedby treatment of the resin with a solution of piperidine indimethylformamide (20%, v/v), respectively. Protected Fmoc-amino acids(NovaBiochem) were activated at their carboxyl groups using 3 equivalent(eq) of amino acid, 3 eqbenzotriazole-1-yl-oxy-tris-(dimethylamino)-phosphoniumhexafluorophosphate (BOP), 3eq N-hydroxybenzotriazole monohydrate (HOBt)and 6 eq of DIPEA (N,N-di-isopropylethylamine). The activated Fmoc-aminoacid was coupled to the free amino terminus of the elongating peptide onthe resin. Completion of the each acylation steps was monitored by theKaiser test. Recoupling was performed if the couplings were incomplete.Myristic acid was coupled in the same manner as amino acid. Thephosphopeptide as the C-terminal amide was cleaved from the resin using94% TFA, 2.5% water, 2.5% ethanedithiol and 1% triisopropylsilane(Aldrich). The crude peptide was purified on a C-4 reverse phasesemipreparative (Vydac 250×4.6 mm cm) column Elution was accomplishedusing a 30 min gradient of 10-100% aqueous acetonitrile containing 0.1%trifluoroacetic acid (TFA) at a flow rate of 8 ml/min. The main fractionwas collected and lyophilised.

Alkaline phosphatase (bovine intestinal mucosa) obtained from Sigma wasused for dephosphorylation of peptide at the serine phosphate. A 10 μlof Peptide solution (0.01 mg/ml) was added to 2 ml of 10 mM HEPES bufferat pH 6.8 and 10 units of alkaline phosphatase added. After allowing10-minute incubation for dephosphorylation calcein liposomes (5 μMlipid) were added and fluorescence intensity recorded using wavelengthsof 490 nm for excitation and 520 nm for emission. Control experiment wasconducted in the absence of alkaline phosphates. FIG. 14A compares thetraces showing substantial dye release (upper curve) in the presence ofalkaline phosphatase compared to low release (low release) in theabsence of enzyme. Thus the enzyme treatment yielded thedephosphorylated peptide resulting in activation of the peptide undermildly acidic conditions 3 μl of 0.1 mg/ml of peptide 12 (table 1) wasincubated for 10 minutes with alkaline phosphatase in 20 μl 10 mMTris-HCL pH 8 buffer. This was then added (see arrow on FIG. 14B) to 2ml PBS pH 6.6 buffer containing 3 μl of calcein liposomes (3 mg/ml lipidconcentration). Fluorescence was recorded as a function of time withexcitation and emission wavelengths of 490 nm and 520 nm respectively. Asimilar experiment was carried out in the absence of alkalinephosphatase. The data in FIG. 14B clearly indicates that the enzymetreatment yielded the dephosphorylated peptide resulting in release ofthe dye by the activation under mildly acidic conditions (curve 1)compared to no or little rate increase in the non-treated (curve 2)sample.

Many tumours are known to have either elevated proteolytic levels or toproduce specific enzymes. A protease-specific sequence for instancecysteine or serine protease sensitive sequences could be attached to thepeptide which render the peptide or complexes inactive and cleavage ofthis sequence along with low pH activates the peptide to releasepayload. There are several protease specific sequences, which could beused in this manner. For instance, peptide substrate sequences cleavedby a prostate specific antigen are known. Other proteolytic sequencesfor enzymes like Elastase, thrombin and endosomal lysosomal enzymes suchas cathepsins B,D,H and L are also known. In addition to this suitableproteases could be targeted to cells or tissues.

pH Arming DNA Delivery

Peptide 13 in table 1 was used to demonstrate cloaking peptide activitywith pH and DNA binding. Calcein liposomes containing (5 μM lipid) wereadded to 2 ml phosphate buffered saline containing the peptide (0.4 μM)and the fluorescence recorded. This was carried out at pH values of 6.6and 7.4. The fluorescence intensity indicative of leakage after fiveminutes was expressed as a % of lysis obtained with 10 μl of 10%Triton-X100. the latter taken to represent 100% lysis. The experimentwas repeated whereby the peptide was pre-condensed with calf thymus DNA(Sigma) at charge ratio to give minimum lysis at pH 7.4. The results inFIG. 15 show that the peptide is activated by acidic pH when no DNA ispresent. It can be concluded that DNA binding results in substantialinactivation of the peptide. Thus the DNA condensate of the peptidewould require both the de-condensation and slightly acidic conditions tocause lysis. Cloaking of peptides with DNA binding and pH has obviousadvantages when delivering genes via the endosome route where low pH isencountered. A combination of highly anionic region (towards the Nterminal end of peptide 13 table 1 residues LEAALAEALEAL) and highlycationic region (the c terminal end of peptide 13 table 1, residuesRRRRQQ,) within the same polypeptide sequence is critical to thisfuction.

To assess condensation of DNA to peptide and its effect on cytolyticactivity, assays were performed at several different pH values and atdifferent peptide to DNA ratios. Peptide appears to be inactive atphysiological pH bound to DNA while activity is regained at acidic pHwhere the DNA is substantially dissociated. The peptide or complexeshave a property of retaining high basic character on one end which isessential for DNA binding while retaining highly acidic character on theother half of the molecule which is critical for pH switching.

Drug Delivery

The peptide was shown to be triggered closer to physiological pH andthis could be used for delivery drugs to acidic areas. The peptide has afatty acid attached which could be used to form complex with drugcontaining liposomes. These liposomes have therapeutic importance. Thepeptide liposome assemblies could be targeted to cells or accumulated inthe tumours whereby binding to a specific marker and a change in pHeffects specific release of the drug. Alternatively, the complexes maytrigger drug release by simple pH change. Delivery to tumours haveclinical relevance as some tumours are shown to be acidic compared to pHof normal tissue. In order to improve delivery of drugs to cancer cellsthe peptide could be inactivated by attaching protease specific sequencewhich would be cleaved in the tumour. However, to control the activityfurther the peptide would then require acidic pH to release liposomecontents. This way damage to any normal tissues which may have traces ofthe same protease present could be minimised by very biospecific releaseat the target site.

Liposomes encapsulating Calcein (120 mM) and with label DiI (the dye tolipid ratio was around 100 μg dye: 100 mg PC) were used. C3H syngenicmice 8-10 weeks old weighing 20-25 g were used. Subcutaneous tumour wasimplanted using KHT cells (5×10⁶ cells/animal) on the dorsal side aftershaving the mice. Typically 200 μl of peptide liposome complex (100 μlof dual dye liposomes+100 μl of peptide 0.1 mg/ml) were administeredintravenously per mice and tumours excised 3 hrs post inoculum. Thetumour was removed aseptically and kept in PBS pH 8.0 buffer and thinsections (160 μm-200 μm) were cut on the slicing machine, and examinedunder a fluorescence microscope. The pH of the buffer was modulated byadding 400 μl of NaP buffer pH 5.8 while simultaneously removing 400 μlof pH 8 buffer. Changes in fluorescence intensity were recorded. Thestate of liposome peptide complex is then determined, whether intact orlysed by measuring fluorescence in tissue slices before-and afterincubation in acidic buffer. The increase in fluorescence in the acidicbuffer indicates the quantity of liposomes that were still intact andable to respond at the time of sacrifice. FIG. 11 shows that theliposome complexes with peptide 9 were able to trigger release ofcalcein in this ex vivo experiment. Upper curve shows fluorescenceintensity of Calcein dye while the lower curve shows fluorescenceintensity of DiI dye. A similar demonstration was then made in vivo asdescribed below.

For the in vivo experiments the tumours were grown in the dorsal skin ofC3H mice. Window chamber measurements were carried out on RIF tumourallografts. To achieve acidification of tumour at the time of liposomeinjection a portion of animals were pre-treated with MIBG/glucose.Tumour bearing mice were given an intra-peritoneal injection of MIBG tolower tumour pH, at a dose of 40 mg/kg MIBG (meta-iodobenzylamine, 0.01ml/g body weight of a 4 mg/ml solution in PBS) and 1.5 g/kg D-Glucose(0.01 ml/g body weight of a 0.15 g/ml solution) given one hour prior toinjection of the liposome preparation. The mice were then given theagent (0.1 ml of liposomes+0.1 ml of PBS pH 8) or peptide-liposomecomplex (0.1 ml+peptide in. PBS pH 8) in total volume of 0.2 ml by atail vein injection. The control or peptide-liposomes were injected intothe tail vein while the mice were on the microscope stage. Thecomputer-controlled imaging system was instructed to begin acquiringtime-lapse images in both fluorescence channels (Calcein and DiI).Images were acquired at a rate of 4 to 12 images per minute. Changes influorescence were monitored continuously. DiI intensity was measuredusing a Texas Red filter set (excitation 560/dichroic cutoff595/emission 620). Calcein intensity was measured using a fluoresceinfilter set (480/5.05/520). The release rate of calcein dye fromliposomes was determined by a dual fluorophore ratiometric method.Automated data acquisition routines were written usingimaging/instrument control software (Metamorph, Universal Imaging).These routines control the operation of the lamp filter wheel andshutter for control of fluorescence excitation, operation of the cooledscientific CCD camera for image acquisition, and analysis of images. Thefollowing calculations are done in real time: subtraction of backgroundintensity levels; calculation of mean, maximum, and variance offluorescence intensity; relative change in intensity for eachfluorescence channel; and ratio of intensities at different wavelengths.Release kinetics are recorded in raw form as intensity versus time foreach of the two fluorescence channels (green/calcein/contents andred/DiI/liposorne). A normalised kinetic plot is obtained by dividingthe contents signal by the liposome signal.

${{Normalised}\mspace{14mu}{Release}} = \frac{\frac{I_{contents}(t)}{I_{contents}\left( t_{0} \right)}}{\frac{I_{lipo}(t)}{I_{lipo}\left( t_{0} \right)}}$Or${{Normalised}\mspace{14mu}{Release}} = \frac{{I_{contents}(t)}/{I_{contents}\left( t_{0} \right)}}{{I_{lipo}(t)}/{I_{lipo}\left( t_{0} \right)}}$

The raw intensity vs time data collected during the first 30 minutesafter injection was converted into a ratio of calcein fluorescence toDiI fluorescence intensity, and normalised so that the ratio immediatelyafter injection (i.e., when the step increase in tissue fluorescenceoccurs) is taken to be 1. Consequently ratios higher than 1 are taken toindicate release of Calcein.

A rapid jump in both DiI and calcein fluorescence was observedcorresponding to the filling of the vascular compartment of the tissue.Subsequently, a slow increase or decrease of DiI fluorescence occurred,reflecting the combined effects of plasma clearance (tending to reduceintensity) and extravasation into interstitial space or uptake intocells (tending to increase intensity). Calcein intensity alwaysexhibited a continued increase, reflecting the release and de-quenchingof calcein from liposomes.

FIGS. 12 shows the normalised change in calcein intensity for the DiIlabelled Control liposomes in untreated (upper) and MIBG/glucose-treated(Lower) tumour tissue.

FIGS. 13 shows the normalised change in calcein intensity for the DiIlabelled Peptide liposome complex in untreated (Upper) andMIBG/glucose-treated (lower) tumour tissue. The peptide used was peptide9 in table 1. MIBG/glucose treatment was administered 3 hours prior toliposomes.

The rate of calcein release, as indicated by the normalised calcein:DiIratio, remained at ˜1.25 or below for the control liposomes and for thepeptide-liposome complex in untreated control tumour. However, the ratioconsistently exceeded 1.5 for complex in MIBG/glucose treated tumour andusually approached 2 or higher. Compared to the other groups, thetumours receiving peptide complex and MIBG treatment exhibit asignificantly higher peak normalised calcein:DiI ratio (p<0.001 byunpaired t-test). The window chamber data provides strong evidence forrelease of payload (calcein) in response to tumour pH. The peaknormalised ratio indicates the change in calcein fluorescence due torelease from liposomes. A value of 1 indicates no change. Compared to avalue of 1.33, a value of 2 corresponds to a 3-fold greater change.

Data shown in earlier example (FIG. 10) used a different peptide(peptide 1) which was shown to trigger closer to physiological pH thanpeptide 9. As illustrated in FIG. (10) significant release of payloadwas noted even in untreated (No MIBG/glucose) mice.

Measurements of tumour pH were undertaken to show that the pH of thistissue was acidic and in the triggering range of complexes. For pHmeasurements, a needle-type combination pH microelectrode in a 20Gneedle was used (tip diameter 0.89 mm; model 818; Diamond General, AnnArbor, Mich.). These pH electrodes contained an internal referenceelectrode. The animals were anaesthetized and pH measured by insertingthe needle tip probe into tumour. For each tissue type, 15 to 20readings were taken. We compared microelectrode pH measurements intumour tissue of untreated and MIBG/glucose-treated mice. The aim was totest whether the in vivo tumour models are acidic and that theMIBG/glucose pre-treatment protocol induces an additional shift towardlower pH. The untreated animals showed mean RIF tumour pH value of 6.8while the treated animals showed pH value of 6.6. We made similarmeasurements in KHT tumours. Again, tumour pH was acidic typically inthe range 6.64 to 6.69. MIBG treatment made little difference in thistumour model.

Multi-Triggering

It is obvious to those skilled in the art that parameters used forswitching peptide activity could be combined to achieve multi-triggeringwhich does not necessarily involve low pH. These parameters also fallunder the scope of our invention. For instance a protease sensitivepeptide could be rendered inactive by binding to another receptor orantibody or ligand binding protein or DNA whereby proteolytic cleavageand freedom from the bound protein is required to achieve activation.Indeed in the case where the peptide is pH active all three parameterswill need to be met before a trigger is evident.

1. A peptide fatty acid conjugate capable of forming a complex with aliposome, the peptide fatty acid conjugate selected from the groupconsisting of SEQ ID NOS:1, 2 and
 6. 2. A composition comprising: apeptide fatty acid conjugate bound to a liposome having a therapeutic ordiagnostic agent encapsulated therein, wherein the peptide fatty acidconjugate is selected from the group consisting of SEQ ID NOS:1, 2 and6; wherein the liposome is coupled to the peptide fatty acid conjugatethrough the fatty acid; and wherein the liposome lyses at a pH from 6.5to less than 7.4, and the composition is non-cytolytic at physiologicalpH.
 3. The composition of claim 2, wherein a diagnostic agent isencapsulated in the liposome.
 4. The composition of claim 3, wherein thediagnostic agent is detectable by a detector selected from the groupconsisting of optical, electrical, electrochemical, magnetic,electromagnetic and acoustic detectors.
 5. The composition of claim 2,wherein a therapeutic agent is encapsulated in the liposome.
 6. A methodof making the composition of claim 2, comprising: reacting a peptidefatty acid conjugate selected from the group consisting of SEQ ID NOS:1, 2 and 6 with a liposome having a therapeutic or diagnostic agentencapsulated therein; and wherein the fatty acid binds to the liposomeand forms the composition.
 7. A method of delivering a therapeutic ordiagnostic agent, comprising administering the composition of claim 2.